Ribosome complexes as selection particles for in vitro display and evolution of proteins

Abstract

The invention provides a method of displaying nascent proteins or peptides as complexes with eukaryotic ribosomes and the mRNA encoding the protein or peptide following transcription and translation in vitro, of further selecting complexes carrying a particular nascent protein or peptide by means of binding to a ligand, antigen or antibody, and of subsequently recovering the genetic information encoding the protein or peptide from the selected ribosome complex by reverse transcription and polymerase chain reaction (RT-PCR). The RT-PCR recovery step is carried out directly on the intact ribosome complex, without prior dissociation to release the mRNA, thus contributing to maximal efficiency and sensitivity. The steps of display, selection and recovery can be repeated in consecutive cycles. The method is exemplified using single-chain antibody constructs as antibody-ribosome-mRNA complexes (ARMs).

Description

BACKGROUND TO THE INVENTIONA current focus of interest in molecular biology and biotechnology is in the display of large libraries of proteins and peptides and in means of searching them by affinity selection. The key to genetic exploitation of a selection method is a physical link between individual molecules of the library (phenotype) and the genetic information encoding them (genotype). A number of cell-based methods are available, such as on the surfaces of phages (1), bacteria (2) and animal viruses (3). Of these, the most widely used is phage display, in which proteins or peptides are expressed individually on the surface of phage as fusions to a coat protein, while the same phage particle carries the DNA encoding the protein or peptide. Selection of the phage is achieved through a specific binding reaction involving recognition of the protein or peptide, enabling the particular phage to be isolated and cloned and the DNA for the protein or peptide to be recovered and propagat...

AI Technical Summary

Problems solved by technology

However, the size of the libraries which can potentially be produced exceeds by several orders of magnitude the ability of current technologies to display all the members.

Thus, the generation of phage display libraries requires bacterial transformation with DNA, but the low efficiency of DNA uptake by bacteria means that a typical number of transformants which can be obtained is only 10.sup.7 -10.sup.9 per transformation.

While large phage display repertoires can be created (17), they require many repeated electroporations since transformation cannot be scaled up, making the process tedious or impractical.

In addition to the limitations of transformation there are additional factors which reduce library diversity generated with bacteria, e.g. certain antibody fragments may not be secreted, may be proteolysed or form inclusion bodies, leading to the absence of such binding sites from the final library.

However, Kawasaki did not reduce the method to practice in these filings and provided no results.

Accordingly, the method was not optimised and he was unaware of the inefficiency of the system as he described it, in particular that due to the method of recovery of mRNA by polysome disruption.

Separate transcription and translation steps were used, and it was stated that the coupled procedure has lower efficiency; however, no data was provided to this effect.

15, which is costly in materials and time.

Thus, it is not known whether the enzyme might be able to pass through adjacent ribosomes, or cause their removal from the mRNA, or only function on mRNA molecules to which only one ribosome was attached.

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